The present invention relates to devices and methods for the elimination of target cells circulating in the blood of a human or animal. More particularly, the invention involves the use of a continuous-flow, extracorporeal device that ablates target cells by utilizing exogenous material to selectively apply energy to target cells before returning treated blood to the body in a continuous blood flow process.
The in-vivo ablation of solid tumors growing in the body is known in the art and the adverse health effects of such tumors have long been recognized. The presence, however, of certain undesirable cell subsets or organisms (e.g. cancer cells, bacteria, or viruses) in the blood of a human or animal can also have deleterious effects on the health of such humans and animals. The present forms of treatment for diseases associated with these blood borne cell subsets are generally systemic, requiring the treatment of the entire circulating blood. While this systemic approach may be necessary to treat the underlying disease, treating the entire circulating blood with a systemic approach is generally undesirable because such systemic approaches affect not only the undesirable cell subsets but also normal cells and tissues.
The identification of circulating cell subsets for diagnostic purposes is a routine clinical procedure (flow cytometry, etc.). These techniques, however, rely on small blood samples taken from the patient, which are then analyzed. These identification techniques are focused on the enrichment and/or extraction of these cells for diagnostic identification or characterization of a disease.
For example, it has been found that the presence of circulating tumor cells (CTCs) in the blood of patients newly diagnosed with metastatic breast cancer is highly predictive of progression-free, overall survival, and is associated with significant prognostic information. The quantification of CTCs may be based on an automated cell enrichment and immunocytochemical detection system (e.g., the CellSearch System, Veridex, Warren N.J.). In this system, circulating epithelial cells are isolated by antibody-coated magnetic beads in a magnetic field (ferrofluid particles are coated with anti-EpCAM antibodies, two phycoerythron-conjugated anti-cytokeratin antibodies recognizing cytokeratins 8, 18 and 19 to specifically identify epithelial cells, an antibody against CD45 conjugated with allophycocyanin to rule out hematopoietic cells, a nuclear dye DAPI to fluorescently label the cell nuclei, and a permeabilization buffer to allow cytokeratin antibodies entry into epithelial cells), and identified using a. semi-automated fluorescence microscope. Cell images are counted as positive if the morphologic features and staining pattern are consistent with that of an epithelial cell (cytokeratin+, DAPI+, CD45−). Besides antibody-based techniques, it is known in the art to use nucleic acid-based techniques such as RT-PCR to identify CTCs through their expression of differentiation markers (cytokeratins 19 and 20, MUC-1, EGFR, Her-2/neu) or oncofetal antigens (beta human chorionic gonadotropin [beta-HCG]).
The significance of circulating cancer cells has been evaluated in clinical studies. In a prospective multicenter study using the Veridex system, CTCs were detected in ca. 70% of metastatic breast cancer patients with a highest count of 1,491 CTCs per 7.5 ml blood. (see Riethdorf, S. et al., Detection of circulating tumor cells in peripheral blood of patients with metastatic breast cancer: a validation study of the CellSearch system, Clin. Cancer Res., 2007. 13(3): p. 920-8.) 61% of metastatic breast cancer patients had >2 CTCs, 47% >6 CTCs per 7.5 ml blood prior to treatment. (see Cristofanilli, M. et al., Circulating tumor cells, disease progression, and survival in metastatic breast cancer, N. Engl. J. Med., 2004. 351(8): p. 781-91.) In a prospective multicenter trial on newly diagnosed patients with metastatic breast cancer, 52% of patients had >5 CTCs at baseline with a worse prognosis than patients with <5 CTCs per 7.5 ml blood. (see Cristofanilli, M. et al., Circulating tumor cells: a novel prognostic factor for newly diagnosed metastatic breast cancer, J. Clin. Oncol., 2005. 23(7): p. 1420-30.)
Engraftment of CTCs in organ systems other than the primary tumor location requires CTCs with clonogenic potential, sometimes referred to as cancer “stem cells.” These cancer “stem cells” have self-renewal capacity similar to normal hematopoietic stem cells and may be capable of forming tumors in subjects. While such cells have not yet been positively identified in the circulating blood of humans having certain forms of cancer, possibly due to, their rare incidence, such theory is consistent with the understanding that stem cells dividing asymmetrically into stem cells and more differentiated cells forming the bulk of the tumor.
Bacteremia is the presence of bacteria in the blood. The blood is normally a sterile environment, but bacteria can enter the bloodstream as a severe complication of infections, during surgery, or due to catheters and other foreign bodies entering the arteries or veins. Bacteremia can have several consequences. The immune response to the bacteria can cause septic shock, which has a relatively high mortality rate. Bacteria can also use the blood to spread to other parts of the body causing infections away from the original site of infection.
Viruses may also circulate in the blood during certain disease states. For example, the viral load of HIV is indicative of disease progression. The reduction in viral load in the blood is one measure of the efficacy of therapy.
Because these types of target cells in the blood generally are rare events and comprise a small number of the total number of cells circulating in the blood stream, most therapeutic techniques focus on the use of drugs to eliminate, inactivate or destroy these cells in-vivo. In the case of cancer cells, for example, chemotherapy or immunotherapies are common techniques. In the case of bacteremia, antibiotics may be used against circulating bacteria. These approaches, however, are not completely effective due in part to increasingly resistant strains of bacteria. Thus, because of the need to avoid significant damage to other circulating blood components and because of the limited number of undesirable cell subsets within the blood, conventional therapeutic techniques are limited in their ability to combat undesirable cell subsets.
Conventional techniques for ablation of stationary in-vivo targets can include the direct application of ablative energy or the use of exogenous materials to transduce the ablative energy at a target site. These techniques can include radiofrequency ablation, thermal ablation using paramagnetic particles activated by alternating magnetic fields, thermal ablation using colloidal metal, plasmonic, or conducting particles activated by electromagnetic radiation, ultrasound based thermal ablation, direct ablation using visible lasers, focused microwave ablation, and similar techniques focused on directing such energy to stationary cell subsets in, for example, a tumor.
Some of these conventional approaches utilized targeting molecules to direct exogenous particles to particular cells or parts of the body. These molecules can be selected from various constructs (peptides, aptamers, antibodies, antibody fragments, and other ligands) that are selective for cell surface receptors on the target cells or that cause the exogenous particle to be internalized by the target cell. The target cell, or the target for the exogenous material, may be an indirect target for ablation, such as endothelial cells of a tumor blood supply. The ablation of these indirect targets may result in the destruction of the ultimate target, such as the tumor itself.
These ablation techniques have comparable methods of cellular elimination that involve the use of light or energy activated molecules that have lethal effect on adjacent cells or tissues. Examples of these comparable methods include photodynamic therapy using photosynthesizers (chemical compound that can be excited by light of a specific wavelength, generally resulting in oxygen radicals). Molecules used for photodynamic therapy include aminolevulinic acid (ALA) and methylaminolevulinate (MAL), among others.
Significantly, the ablative techniques described above are all in-vivo and require energy to be applied to an area of treatment for a specified time, which can range from several seconds to hours. Additionally, most techniques ablate all material within the field of application (e.g. radio frequency ablation, photodynamic therapy, direct laser ablation), thereby resulting in damage to non-target or healthy cells. The use of exogenous energy transducers in these in-vivo procedures allows more specific ablation of solid tissue and tumors. For example, gold nanoshells, comprised of a silica core surrounded by a gold shell, have been designed to absorb near-infrared laser energy. When delivered intravenously to solid tumors, these particles may be activated with a near-infrared laser to thermally ablate the tumor while in the body. In the same vein, the use of targeting ligands with such, nanoparticles may allow an increased level of selectivity of ablation by directing the particle and the applied energy to specific types of cells or a location within the body. U.S. Pat. Nos. 6,344,272 and 6,685,986 teach the compositions and synthesis of one class of nanoparticles. U.S. Pat. No. 6,530,944, which is hereby incorporated by reference, describes localized in-vivo treatments by localized induction of hyperthermia in a cell or tissue by delivering nanoparticles to said cells or tissues and exposing the nanoparticles to an excitation source under conditions where they emit heat. This treatment is applicable to a stationary solid tumor mass.
Other nanoparticles have been described for the in vivo ablation of solid tumors and tissues. For example, paramagnetic particles, gold nanorods and carbon nanotubes have been described, generally with targeting ligands, for the ablation of solid tumors and tissue. These particles have been delivered intravenously or by direct injection into the tumor. These particles may also be delivered through absorption in tumor-targeting cell subsets such as tumor infiltrating lymphocytes. These techniques are applicable to solid tumors.
These techniques have not generally been useful for the ablation or elimination of cells that circulate in blood. These circulating cells either move through an applied energy field too rapidly to allow therapeutic effect or the energy field may not be applied in a manner that can be applied to such cells. For example, visible and near infrared electromagnetic energy have limited depth of penetration through tissue or vessel walls, limiting depth of penetration into the body or blood. Thus, in-vivo activation techniques suffer from the problem of shielding by the body, preventing direct access to the circulating cell subsets. Forms of energy that have greater penetration depth often have undesirable side effects. For example, alternating magnetic fields can result in eddy effects or activation of paramagnetic particles that have cleared from blood but not yet cleared from the body, resulting in adverse effects on healthy tissue.
For the foregoing reasons, conventional techniques for the in-vivo ablation of stationary target cells have many drawbacks for certain applications.
Other conventional techniques, such as the techniques taught in U.S. Pat. No. 6,685,730 use exogenous materials for the purpose of enhanced tissue repair. Such techniques, however, do not teach selective destruction or damaging of cells.
Various extracorporeal devices have been incorporated in other biological processes and methods. For example, dialysis or membrane separation of blood components is a common medical procedure. These techniques are not designed for the treatment of specific cells in the blood, but rather provide for the removal of proteins and molecules normally removed by properly functioning body organs. Apheresis of proteins has also been described for the treatment of diseases, such as dry macular degeneration. These techniques do not treat cells, much less specific targeted cells, during the process.
U.S. Pat. Nos. 4,321,919, 4,398,906, 4,428,744, and 4,464,166, and 5,984,887 describe extracorporeal photopheresis, wherein blood is removed from the body and treated with ultraviolet light and drugs that become active when exposed to such light. The blood is then returned to the body. This technique is being studied in the treatment of some blood and bone marrow diseases (e.g., cutaneous T-cell lymphoma) and graft-vs-host disease (GVHD). In these techniques, mononuclear blood cells are collected by apheresis, treated ex vivo with psoralen, exposed ex vivo to UV light, and finally retransfused to the patient. These techniques are characterized by a batch process (i.e. not a continuous process), because of the nature of the therapy and the length of treatment required, which can last several hours.
Blood warming devices are known in the art and have been used for a variety of purposes. For example, during transfusions, the blood is heated to avoid adverse effects to the patient receiving the transfusion. Following hypothermia, devices have been investigated to heat blood for reinfusion into the patient to elevate body temperatures.
Similarly, extracorporeally elevating the temperature of blood has also been investigated in the treatment of HIV, Kaposi's Sarcoma, cancer and other disorders. Blood was taken out of the body, heated, and then allowed to cool before being returned into the body. Additionally, investigations of whole body hyperthermia for the treatment of cancer have included extracorporeal heating of blood prior to reinfusion to elevate body temperature.
None of the foregoing techniques, however, are useful for targeting specific undesirable cells in the blood.
Likewise, apheresis and similar techniques are known in the art. For example, U.S. Pat. No. 6,528,057 describes a method for reducing viral load by removal of viruses or fragments or components thereof from the blood by extracorporeally circulating blood through hollow fibers which have in the porous exterior surface, immobilized affinity molecules having specificity for viral components. Passage of the fluid through the hollow fibers causes the viral particles to bind to the affinity molecules so as to reduce the viral load in the effluent.
U.S. Pat. No. 5,104,373 describes a method for extracorporeally treating blood samples by one or all of several modalities, including (i) the hyperthermic treating of blood at a reduced pH; (ii) mechanically damaging or lysing blood cells that contain or have been affected by a virus, microorganism or disease state, and so as to render more fragile than other cells; and (iii) subjecting the blood to irradiation. This device, however, is not selective in its application of irradiation to the cells in or components of blood of the patient. Disadvantages of these conventional techniques include failing to preferentially treat the undesirable cell subsets in the irradiated blood stream as opposed to treating the entire irradiated blood stream.
United States Patent Publication No. 2004/0191246 describes a device for the separation of biological cells. The application describes the use of the separated cells for immunotherapy and other means by the in-vivo treatment of bodily fluid, and also makes reference to the “neutralization” of such cells, but does not describe the methods for such neutralization, nor does it describe how such methods distinguish between the target and the remaining blood cells. Additionally, this application contemplates separating the targets from the remaining blood components within the device.
Various devices have been developed for the separation or enrichment of cells from samples of body fluid, yet these devices are not limited to operating on only the sample itself and do not teach treating the entire blood component of a patient. U.S. Patent Publication No. 2006/0252087 describes methods for the separation of cells or target molecules from a body fluid sample. U.S. Patent Publication No. 2006/0141045 describes beads that may be used for cell separation from body fluid samples. U.S. Patent Publication No. 2007/0161051 describes a device with a similar function. Other examples are also described in the literature. These devices, however, are designed to utilize a small fluid sample and therefore are not useful for treating the entire blood volume of a patient.
Accordingly, improved methods are needed that address one or more disadvantages of the prior art.